Enzymes In Detergents

Transcript

Page | 1 AMITY INSTITUTE OF BIOTECHNOLOGY ENZYMOLOGY ASSIGNMENT-I ENZYM ENZYME ES USED IN DETERG DET ERGEN ENTS TS DATE DA TE OF SUBMISSION- 22.O4.08 SUBMITTED SUBMI TTED TO: SUBMITTED SUBMIT TED BY: BY: DR. S.M. S.M . BHATT BHATT MAYANK MAYANK JAIN JAI N FACULTY ACULTY,ENZYMO ,ENZYMOLOGY LOGY ROLL NO.-77 AIB SECTION-U AIB CONTENTS: CONTENTS : HISTORY................................................................................. INTRODUCTION................................................................... PAGE NO. 3 3 DETERGENT ENZYMES PROTEASES....................................................................... 5 AMYLASES......................................................................... 7 LIPASE................................................................................ 8 CELLULASE...................................................................... 9 Page | 2 MISCELLANEOUS DETERGENTS PEROXIDASES................................................................. 10 PULLULANASE................................................................ 10 ENZYME FORMULATION............. FORMULATION........................ ....................... ....................... ............... .... 11 PRODUCTION OF ENZYME BASED DETERGENT........ DETERGENT........ 13 ENZYME STABILITY STABILITY.......... ..................... ....................... ....................... ...................... ................. ...... 14 APPLICATION APPLICATION OF ENZYME BASED DETERGENT........ DETERGENT........ 16 BENEFIT OF USING ENZYME IN DETERGENT.......... DETERGENT.............. .... 19 CONCLUSION ...................... ........... ...................... ....................... ....................... ...................... .................. ....... 20 BIBLIOGRAPHY....................................................................... 22 History The original idea of using enzyme as detergents was described in 1913 by Dr  Otto Rohm, who patented the use of crude pancreatic extracts in laundry presoak compositions to improve the removal of biological stains. In the same year, the first enzymatic enzymatic detergent named Burnus was launched, launched, but was not popular   because of its own limitations. Subsequently, Bio- 40 - a detergent containing a  bacterial protease was produced in Switzerland and launched in the market in 1959 and it gradually became popular. In the period from 1965 to 1970, use and sale of detergent enzymes grew very fast. In 1970, the use was distorted due to dust production by formulations leading to allergies to some workers. This  problem was overcome in 1975 by encapsulating the granules of enzyme. From 1980s to the 1990s, several changes took place in the detergent industry like deve develop lopme ment nt of soften softenin ing g thro throug ugh h the wa wash sh,, deve develop lopme ment nt of conc concen entra trate ted d heavy-duty power detergents, development of concentrated or structured or nonaqueous liquid detergent. Introduction Enzymes have been used to improve the cleaning efficiency of detergents for  more than 35 years, and are now well accepted as ingredients in powder and liquid detergents, stain removers/laundry pre-spotters, automatic dishwashing deterg detergents ents and industr industrial/ ial/inst institu itution tional al clea cleanin ning g produc products. ts. Det Deter ergen gentt enzyme enzymess account account for about 30% of the total worldwide worldwide enzyme production and represent Page | 3 one one of the the larg largest est and and most most succe successf ssful ul appl applic icati ation onss of mode modern rn indus industri trial al  biotechnology. The largest segment within the global industrial enzyme market is the market for technical enzymes, estimated at around uss 980 million in 2002. In the technical enzymes category, detergent additives make up for nearly two-thirds of the market. These enzymes are used as functional ingredients in laundry dete deterg rgen ents ts and and autom automate ated d dishw dishwash ashing ing deter deterge gents nts.. This This artic article le gives gives an overview of the detergent enzymes industry and discusses its manufacturing and downstream processing. Enzymes used in detergents are protein catalysts that consist of long chains of  amino acids. They are similar to protein catalysts present in all living cells wher wheree they they contr control ol metab metabol olic ic proce processe sses, s, conv conver ertt food food nutr nutrien ients ts to simpl simplee molecules, convert these molecules to energy and to new cell material. As catalysts; enzymes speed up specific chemical reactions, in mild conditions of  temp temper erat atur uree and and pH, pH, wi with thou outt bein being g alte altere red d or cons consum umed ed in the the proc proces ess. s. Cons Conseq eque uent ntly ly,, smal smalll quan quanti titi ties es of enzy enzyme me ca can n repe repeat ated edly ly ca cata taly lyze ze the the  breakdown of millions of molecules in minutes. Enzymes function optimally in detergents at temperatures of 20 - 60C and within a pH range of pH 7.5 - 10.5. The The perfo performa rmanc ncee of enzy enzyme mess in deter deterge gents nts depe depend ndss on numbe numberr of factor factors, s, inclu includi ding ng the dete deterg rgen ent’ t’ss compos compositi ition on,, type type of stains stains to be remov removed ed,, wa wash sh temperature, temperature, washing procedure procedure and wash-water hardness. To help formulators formulators optimize enzymatic detergent washing efficiency, Specialty Enzymes provides wash laborat laboratory ory tec technic hnical al service services. s. In our was wash h labora laboratory tory,, custome customerr, base base detergents are evaluated on standard soils in both a model wash system (Terg-OTometer) and in full-scale household washing machines. Table 1 Compositions of an enzyme detergent det ergent Constituent Composition (%) Sodium tripolyphosphate (water softener, loosens dirt)a 38.0 Sodium alkane sulphonate (surfactant) 25.0 Sodium perborate tetrahydrate (oxidising agent) 25.0 Soap (sodium alkane carboxylates) 3.0 Sodium sulphate (filler, water softener) 2.5 Sodium carboxymethyl cellulose (dirt-suspending agent) 1.6 Sodium metasilicate (binder, loosens dirt) 1.0 Bacillus protease (3% active) 0.8 Fluorescent brighteners 0.3 Foam-controlling agents Trace Page | Perfume Water 4 Trace to 100% Detergent Enzymes Pres Presen entl tly y, dete deterrgent gent enzy enzyme me has has beco become me an inte integr gral al part part of dete deterrgent gent formulation. A look at the market share of detergent enzyme indicates it to be very high in comparison with other enzyme applications. Enzymes that have to  be used as detergent composite must possess the following characters:     Stability at temperature over a broad range of 20C to 50C and even above The optimum pH should be in alkaline or higher alkaline range It should be detergent compatible It should have specificity towards different proteins amylases, es, lipas lipases, es, cellul cellulases ases,, Major Major deterg detergent ent enzyme enzymess include include proteas proteases es, amylas miscellane miscellaneous ous enzymes enzymes such as peroxidase peroxidasess and pullulanase pullulanase. A recent trend is to redu reduce ce this this phosp phospha hate te cont conten entt for for envir environm onmen enta tall reaso reasons ns.. It may may be replaced by sodium carbonate plus extra protease. Proteases Proteases were introduced in the market in 1959 in the detergent Bio-40,  produced by Schnyder Ltd in Switzerland. Most powder and liquid laundry detergents in the market, today, contain proteases. Proteases are of two types:  Alkaline protease from Bacillus licheniformis, having optimum pH 8, for  egg, liquid laundry product, (pH 7- 8.5), commercially known as Alcalase -Novonordisk Optimase- Genencor Inter . Page |  5 High alkaline protease from Bacillus alkalophilus and Bacillus lentus, having an optimum pH 10. For e.g., powder laundry products, automatic dish washing formulations, known by trade names of Savinase-Novo   Nordi Nordisk, sk, Purafet Purafet-- Genenc Genencor or Inter Inter. Proteas Proteases es enhanc enhancee the clea cleanin ning g of   protein-based  protein-based soils, such as grass and blood by catalyzing the breakdown of the constituent proteins in these soils through hydrolysis of the amide bonds between ind individu idual ami amino acids ids. In the the ca casse of serin rine endopeptidase, it contains a catalytic triad of amino acids at the active site; • An aspartyl residue containing ß-COO¯  • A histidine containing the imidazole group • A serine residue with p-OH as the functional group The serine hydroxyl group functions as a potential nucleophile, where as both the aspartyl and histidine functional groups behave as general base catalysts facilitating the hydrolysis process. . The serine group initiates the nucleophilic attack on the peptide bond to form a tetr tetrah ahed edra rall inte interm rmed edia iate te,, whic which h unde underg rgoe oess an ac acti tive ve hydr hydrog ogen en tran transf sfer er,, facilitated by both the histidine and aspartyl residues. The net effect of the addition of water across the bond generates the original protein. The protease hydrolysis involves the transfer of electrons between the amino acids at the active site and substrate. For proteases the three-dimensional arrangement of the catalytic triad is required for the enzyme to be active. Disturbances in the confi confirm rmati ation on are likely likely to affe affect ct enzy enzyme me effic efficac acy y and and theref therefor oree cle clean anin ing g  performance.    These were susceptible to oxygen bleaches and calcium sequestrates. But now, stable protease can be obtained . Oxidative attack by peroxides or per acids on the methionine residue adjacent to the catalytic serine results in nearly 90% loss of enzyme activity. However, replacing methionine with oxidatively stable amino acids like alanine improves stability of enzyme towards oxygen bleach (Boguslawski et al, 1992) Protease substilisin requires at least one calcium ion, which maintains three- dimensional structure of enzyme. However, calcium- sequestering agents used in many laundry procedures to control water hardness can remove this calcium resulting in the decreased thermal and autolytic Page | 6 stability stability. This can be corrected corrected by the introduction introduction of negatively negatively charged resid residues ues near near the calci calciumum-bi bind nding ing site, site, which which incre increase asess the bindi binding ng affinity of enzyme for calcium and results in improved stability towards calcium sequestrants (Krawczyk et at, 1997)   Protease has limited applications towards the detergency of wool and Constituent silk, because of the proteinaceous nature of these fibres. Proteases are added in an encapsulated or granulated form, which protects them them from from other other dete deterg rgen entt ingre ingredi dien entt and and elimi elimina nates tes the the probl problem em of  auto autoly lysi siss or prot proteo eoly lysi siss of othe otherr enzy enzyme mes. s. In aque aqueou ouss dete deterrgent gent formulations, protease inhibitors show a preventive effect of avoiding contact of the protease molecules with each other as well as other enzyme molecules. This effect gets nullified on dilution and enzyme molecules are free to act on stains (Krawczyk et al, 1997) Amylases Amylases facilitate the removal of starch-based food soils, by catalyzing the hydro hydroly lysi siss of glyc glycos osidi idicc linka linkage gess in starc starch h poly polyme mers. rs. Ge Gene nera rally lly,, starc starchhconta contain inin ing g stain stainss are of choco chocola late te,, gravy gravy,, spagh spaghett etti, i, coco cocoa, a, pudd puddin ing, g, etc etc.. Amylases can be classified as: : a-amylases: These enzymes catalyze the hydrolysis of the amylose fractions of  the starch under hydrolysis of the glycosidic bonds in the interior of the starch chai chain. n. The The firs firstt step step in the the reac reacti tion on is ca call lled ed as endo endore reac acti tion on & lead leadss to oligo oligosa sacch cchar aride ides, s, wher wheree short short chain chain wa wate terr- solub soluble le dext dextri rins ns are are produ produce ced. d. . ß-amylases: Thes Thesee enzy enzyme mess ac acto ton n dext dextri rins ns from from redu reduci cing ng end end and and form formss maltose units. Pullulanases or isoamylases: These degrade degrade starch directly directly into linear dextrins for for they they also also attac attack k ci-1 ci-1,6 ,6 glyco glycosid sidic ic bond bonds. s. . . Amyloglycosidases: These enzymes act on the dextrin or maltose units and forms glucose units. . a-amylases are mostly used for detergents, although recently other carbohydrate cleaving enzymes such as pullulanases or isoamylases have also been described Page | 7 for this application. a-amylases bring about the primary hydrolysis of starch into the oligosaccharides and dextrins. Currently, these enzymes are produced from   bac bacte teri ria. a. Baci Bacill llus us subt subtil ilis is.. Baci Bacill llus us amyl amylol oliq ique uefa faci cien ens, s, and and Baci Bacill llus us licheniformis. These are available under the trade names Maxamyl- Genencor  Int or Termamyl -Novo Nordisk  Lipases Tomato-based sauces, butter, edible oils, chocolate and cosmetic stains are very difficult to remove as they form due to greasy food stains. Body soils, sebum and sweat on collars, cuffs and underarms, are generally composed of a mixture of proteins starch pigments and lipids. Lipases hydrolyze the water insoluble trig riglyc lycerides comp omponents into the more water-sol soluble products as monoglycerides, diglycerides, free fatty acids and glycerol. The Novo Nordisk  launched the first lipase product in 1987. They transferred the lipolase gene into the fungus Asper6yillus Asper6yillus oryzae for industrial production, Genencor followed in 1993 with lumafast (Pseudomonas menocina) and Gist-Brocades in 1995 with Lupomax. . Currently, the known sources of lipases include mammalian lipases (human  pancreases/colipases), fungal (Rhizomucor mehei, Humicola lanuginose, etc), yeas yeastt (Can (Candi dida da rugos rugosa, a, Cand Candid idaa anta antarti rtica ca), ), bacte bacteri rial al lipas lipasee (Pse (Pseud udomo omona nass glumae, Pseudomonas aeroginosa, Chrobacterium viscosum) (Ishida et al, 1995) . . Lipases possess a catalytic triad that is similar to the serine proteases of trypsin and subtilisin type. Hence, these are also called as serine hydrolysate lysate. Lipases can decompose a fatty stain up to 25%, which then can be removed very easily because of the hydrophilic character (Dorrit et al, 1991). It is generally thought that lipases get adsorbed on to the hydrophobic stain during the washing period. And, during the drying cycle when the water content is decreased, the enzyme is activated and can hydrolyze triglycerides in the stain. This facilitates the removal of stain in the next wash cycle (Dorrit et al 1991). The enzyme also has stability over a broad range of temperature 30C to 60C. These novel alkaline lipases also retained 100% activity in the presence of  strong oxidants. Effo Effort rtss are are on to manuf anufac actu ture re enzy enzyme mess that that ca can n work work belo below w the the norm normal al temperature range of 30C to 40C to save energy. It has been observed that energy consumption per wash in household washing machine (3 kg clothes) at low low tem tempera peratu ture re (30C (30C)) is less less than than 1 % of the the ener energy gy used used at a high higher  er  temperature (60C) (Edvardetal, 1991). . Page | 8 Cellulases These enzymes introduced in the late 1980s were described for the first time in a Japanese patent filed by Murata. These enzymes are used in UK and US since 1991. Cellulases remove microfibrils from cotton and cotton-blended fabrics. These microfibrils stick out from the main fibre of cotton and are formed during use and repeated washing condition of the tissue. This makes garments and household textiles unusable. The cellulases can be used as softening agents, to remove soil particles and to revive colours. . . The overall cellulose structure has two types of region; one that has a higher  orde orderr of cryst crystall allin inity ity is calle called d crys crystal tallin linee regio region. n. The The othe otherr type type has has less less structured order, and hence is called as amorphous regions. The activating but not hydrolytically acting enzyme was named as C i-activity. According to this concept, concept, microorgani microorganisms sms that are able to degrade crystalline cellulose have C iactivity. This enzyme is not present in that microorganism that attack only substituted cellulose like carboxymethyl cellulose as they have Cx-activity. Accord Acco rding ing to a rece recent nt rese researc arch, h, biode biodegr grad adati ation on of cellu cellulo lose se requi require ress the the interaction of three different hydrolytes or at least the first two enzymes to attack simultaneously. These include: · Cellobiohydrolase is also also ca call lled ed as exoc exocel ellu lula lase se (C i-ac i-acti tivi vity ty)) . . · Endoglucanases is also calle lled as Endoce ocellulas lases (C i-ac -activity ity) . . · ß-Glucosidase is also called as cellobiase ase . . It was observed that sebum in the interior of cotton fibres cannot be removed by ordinary detergents satisfactorily, although they readily remove sebum on the exterior of the fibres. Alkaline cellulase interacts selectively with cellulose in interfiber interfiber spaces in the interior of fibre, and selectively selectively removes the sebum soil. The removal of the soil is by the hydrolysis of amorphous regions (Murata et al, 1991). Cellulases can be chemically modified to have greater stability and efficiency in alkaline medium. It can be done by treating the acid cellulases with reagents like maleic anhydride (Bund and Singhal et al, 2002). Page | 9 . Currently Currently,, the cellulases used in detergents detergents are manufactured manufactured from bacteria bacteria and fungi. Bacterial cellulases have been in use since 1987, for example, Biotex. Som Some gene geneti tica call lly y engi engine neer ered ed stra strain ins, s, which hich are are wi wide dely ly used used incl includ udee Strep Streptom tomyc yces es sp. sp. KS KSM-2 M-2,, Baci Bacillu lluss KS KSM-6 M-635 35.. The The fung fungal al cellu cellulas lasee from from Humicola isolens DSM1800 is active under mild alkaline conditions. Miscellaneous detergent enzymes Peroxidases: These are one of the newest classes of enzymes that have been included in detergent formulations. Peroxidases are subclass of general oxidoreductases and are very popular and commercially available for manufacturing detergents.  Novo Nordisk produces this under the brand name Guardzyme obtained from mushroom Corprinus cinereus. It is a heme containing protein, which in the  presence of H2O2 can mediate the oxidation of fugitive dyes in solution and inhibits the dye transfer. Pullulanases: In recent years, pullulanases (Pullulan 6- glucanohydrolase) a debranching enzyme has been gaining importance due to its efficiency of starch hydrolysis  by cleaving a-1, 6 linkages. Pullulanases with other amylolytic enzymes are used in detergents for improved stain removal and enhanced overall cleaning  performance. This enzyme was first isolated in Klebsiella pnuemoniae (Shaw et al, 1995). Manufacturing and downstream processing  Nearly all-detergent enzymes, which are used and marketed today, are produced through large-scale fermentation of microorganisms. Most of enzymes "are obtained from the bacterial or fungal strains. As low cost enzymes are needed to support the requirements of the global detergents business, enzyme manufacturers should consider the following points to ensure lower costs: Page |    10 The enzyme-producing micro-organism must be capable of secreting the enzyme extra-cellularly in the bulk fermentation broth, as the cost in terms of both money and time to recover enzyme from the fermentation  broth is very high The production organism should be able to produce highest possible yields. Strain optimisation can be accomplished either through classical mutagenesis and screening methods or using genetic engineering · The number of steps in the downstream processing should be kept to a minimum to be economical and also to avoid yield losses The production organism should produce the desired enzyme in a highly  pure state without any contaminating side activities or proteins. This can  be done by deleting the genes, which codes for unwanted enzymes and  proteins Enzyme formulations . Enzymes are formulated mainly in two forms, as a liquid product or as a granular product . 1. Liquid product formulation . The highly concentrated liquids of the evaporator or the ultra filtration unit can be used for the manufacturing of the liquid formulations. The liqui liquids, ds, whic which h are are to be inco incorpo rporat rated ed into into the the formu formulat lation ion,, must must be sterilized against microbial growth. Stabilizing agents like borax, organic   bor boric ic acid acid deriv derivat ative ives, s, alka alkali li salts, salts, etc etc shoul should d be adde added d along along wi with th  preservatives like urea, propel glycol, diglycol, and sorbitol. The current trend is to formulate these liquid formulations as structured liquids with the help of salts and polymers, so that all surfactant remains in the structured structured liquid and enzymes enzymes remain remain in the aqueous phase. (Hermann (Hermann et al, 1997) 2. Granular enzyme products . Highly High ly ultr ultraa filt filter ered ed and and dial dialys ysed ed enzy enzyme me solu soluti tion on is subj subjec ecte ted d to adjustment of pH, turbulences, and temperature in the suitable range, when the enzyme crystallises out. It can be precipitated at high salt conc concen entra tratio tions ns.. The The follo followi wing ng four four type typess of granu granula latio tion n proc process ess are are employed: i. Enzyme pulling: : The enzyme (dry) is dispersed into a molten wax, non-ionic surfactant, or polymer matrix, and then sprayed in a cooling tower to form solid, spherical, molten water-soluble or water dispersible material with a Page | 11 melting point above 50C. This technique offers the advantages of high throughput and ability to recycle particles that fall outside the desired size range but has a drawback i.e., the particle has relatively poor   physical strength, leading to break- up and high dust generation in subsequent processing. Polyethylene glycols can be used to improve  physical  physical strength and thus lesser dust formation, formation, but as particle breakup cannot be completely ruled out, it is not used widely at present. ii. Granulation by extrusion process: : In this technique, all the ingredients like enzyme powder or liquid conc concent entra rate te wi with th bind binder ers, s, such such as clay clay suga sugarr, starc starch, h, some some antianticloggin clogging g agents agents like cel cellulo lulose se fibres fibres and solubili solubility ty enhanc enhancers ers like sodium sodium sulp sulpha hate te are are mixed mixed toge togethe therr and and an extr extrud udab able le doug dough h is   produced, which is then pressed through the perforated metal plate. The extruded noodles are cut into small cylinders and then given a round shape by a spheroniser. spheroniser. After sieving, the particles particles are coated with pigments such as titanium dioxide, and protective outer layers to achieve desired appearance and to improve granulate integrity. The drawback drawback of this method is the high capital investment investment in a multi-step   proc process ess and and the sensi sensitiv tivit ity y of the the proce process ss varia variatio tion n in feeds feedsto tock  ck  moisture and composition. iii. High shear granulation: : In this process the enzyme is mixed with controlled amounts of water,  binders such as polyethylene glycol, ethoxylated fatty alcohols, fatty acids, bentonite, waxes having low melting temperature and other  granulating agents so as to form a low-moisture agglomerate. This agglomerate is then passed on to the high-shear mixer in which it is  broken up into smaller particles. The particles are then dried in a fluidised bed and coated with a final protective layer and pigments like titanium dioxide. . iv. iv. Fluid bed coating : : In this process, on the inert support or core material like sodium chloride, calcium alginate, urea, or saccharose beads, liquid enzyme is sprayed and the coating material is transferred transferred to the drying zone with the help of heated air stream. Then once the enzyme layer has dried, additional coatings of stabilizers, chelating agents, antioxidants and   pigm pigmen ents ts are are appli applied ed.. The The outer outer coati coating ng consi consists sts of film film formi forming ng   polymer such as titanium dioxide. The volume of flow for proper  fluidization is dependent on the surface area and the shape & density of the core material. The proper fluidization should flow sufficient core material through the spray zone to coat all the atomized liquids Page | on . to the core material to prevent spray 12 drying. Production Production of enzyme-based detergents The manufacture of an enzyme detergent is not very different from that of the conven convention tional al synthe synthetic tic one. one. Modern Modern indust industrial rial cultiva cultivation tion of enzyme enzymess begins begins with wi th ferm fermen enta tati tion on of a vial vial of drie dried d or froz frozen en micr microo-or orga gani nism smss ca call lled ed a  production strain. This production strain is selected to produce large amounts of  the enzymes of interest. The production strain is first cultivated in a small flask  containing nutrients and agar. The flask is placed in an incubator which provides the optimal temperature for the previously frozen or dried cells to germinate. Once the flask is ready, the cells are transferred to a seed fermenter, which is a large tank containing previously sterilized raw materials and water, known as the medium. Seed fermentation allows the cells to reproduce and adapt to the environment and nutrients that they will encounter later on. The cells are then transferred to a larger tank, the main fermenter, where temperature, pH and dissolv dissolved ed oxygen oxygen are careful carefully ly controll controlled ed to optimi optimize ze enzyme enzyme produc production tion.. Addi Additio tiona nall nutr nutrie ients nts may may be adde added d to enha enhanc ncee prod produc uctiv tivity ity.. When When main main fermentation is complete, the mixture of cells, nutrients and enzymes, referred to as the broth, is ready for filtration and purification. The prime step for the formulation of an enzyme-based detergent is the compatibility of the enzyme(s) with various detergent ingredients. In general, the suitability of an enzyme   pre prepa para rati tion on main mainly ly depe depend ndss on its its comp compat atib ibil ilit ity y wi with th the the dete deterg rgen ents ts at moderately higher temperatures. An ideal enzyme for detergent preparation should be effective at low levels (0.4–0.8%) in the detergent solution. It should also be compatible with various detergent components along with oxidizing and sequestering agents and possess adequate temperature stability to be active in a wide range of cleaning temperatures. It must also have a long shelf life11. Moreover, the very low use concentration is due to the fact that the enzymes added to the product are biocatalysts. In this context, the term biocatalyzation is implied wherein the enzymes themselves are not being consumed during the cleaning cleaning process and a single enzyme triggers numerous chemical chemical reactions. As a result, the disadvantages of the conventional detergents are eliminated. The early use of enzyme powders in detergents led to dust problems in the  production process. In addition, the reduced stability of the enzymes due to autol autolys ysis is and and detr detrime iment ntal al effec effects ts by the the other other dete deterg rgen entt ingre ingredi dien ents ts in the   pre presen sence ce of moist moisture ure we were re enco encoun unter tered ed.. Thes Thesee prob problem lemss led led to the use use of  granulation techniques and enzyme prilling with enzymes being encapsulated in an inert water soluble waxy substance. In powder detergents, the enzymes are mixed with the finished powders as granulates or prills. Currently, wax-coated enzyme detergent granules are being offered in colours identical to the non- Page | 13 colou coloure red d deter deterge gent nt gran granule ules. s. The The colou coloure red d gran granule uless are are terme termed d as  signal   granules by the detergent manufacturers which symbolize the presence of an extra-added active ingredient in the detergent preparations. In case of enzyme-based automatic dishwashing detergents, citrate and other    polyacrylate builders are added. Moreover, perborates and percarbonates are also also used. used. These These pero peroxyb xyble leac ach h gene generat rating ing syste systems ms are are not not too too harmf harmful ul for  enzym enzymes es and and throu through gh the actio action n of activ activat ators ors such such as tetra tetraac acety etyle lethy thyle lene ne diamine (TAED), enable the acceptable bleaching action at low temperature. Enzyme stabilization Most early enzyme products such as detergent proteases were just powders. Almost all of them were granulated and further protected by coatings. Another  method to prevent enzyme dust in the air is liquid formulations. Today a lot of  research work is being done in the different formulations and stabilization techniques in many enzyme detergent production facilities. The enzymes used in various detergent formulations are subject to proteolytic and autolytic degradation on storage and sudden exposure to harsh operating conditions results in rapid inactivation of enzyme activity. Loss of enzyme activity is also encountered during storage in the factory, shipment to client(s) and/or storage in client(s) facilities. Hence, storage stability is of prime concern to enzyme manufactures. The rate of enzyme inactivation is largely dependent on temperature, pH and other detergent components such as surface active agents, sequestrants and bleaching agents. Moreover, the higher the temperature and alkalinity, the less stable is the enzyme. The loss of the enzyme activity is mainly due to the partial unfolding of the  polypeptide chain, since the inactivating agent breaks down the delicate balance of nonco noncova vale lent nt bonds bonds whic which h main maintai tain n the nativ nativee confo conform rmat ation ion.. The The ideal ideal appr approac oach h to stabil stabiliz izee the the enzy enzyme me would would be to iden identif tify y the mecha mechanis nism m of  inactivation and then design a procedure which would prevent that mechanism. In order to protect the enzyme against denaturation, addition of stabilizers like calc ca lciu ium m salt salts, s, sodi sodium um form format ate, e, bora borate te,, poly polyhy hydr dric ic alco alcoho hols ls and and prot protei ein n   prepa preparati rations ons have have proved proved successf successful. ul. To preven preventt contam contaminat ination ion of the final final commercial crude preparation during storage, addition of sodium chloride at 18–2 18–20% 0% conce concent ntra ratio tion n has has been been sugg suggest ested ed.. These These proce process sses es maint maintai ain n the enzyme activity and improve storage stability. In certain cases, for the purpose of convenience in handling and storage, liquid enzyme preparations are often brought to powder form by vacuum or air drying which are milder and less expensive than lyophilization. The stabilization of enzymes has also been made possible through use of protein engineering to design tailor-made enzymes with specific enzyme properties and stability and and this this tech techni niqu quee is lead leadin ing g new new insi insigh ghts ts into into the the proce rocess ss of   biocatalysis. Protein engineering is rapidly emerging today as a new science and Page | 14 is basically an art of modifying an existing protein or creating de novo, a protein of pre-specified properties. From a commercial viewpoint, this technology is inhe inhere rent ntly ly comp comple lex, x, cost costly ly and and time time cons consum umin ing. g. De Desp spit itee thes thesee inhe inhere rent nt drawbacks, commercial detergent enzyme producers adopt this technology for   producing novel and/or superior enzymes with stable, new and/or improved   properties like stain removing ability, improved stability due to resistance to oxidizing agents (oxygen-based bleaching), etc.. Applications of enzyme-based detergents Data published up to now indicate that the enzyme detergents are being mainly explored for their application in laundry, dishwashing, textile and other such industries. industries. Of late, in view of their advantages and increased potentiality potentiality,, some researchers have tried to use them in the food and dairy industries. The different applications wherein the enzyme detergents are being currently used are: In laundry The microbial enzymes which have found application so far in laundry are the   proteases, amylases and lipases. More recently, the cellulases have also been employed in the detergent industry with an added dimension. The proteases hydrolyse the proteinaceous residues of blood, egg, grass and sweat to form soluble peptides which are subsequently easily removed by detergent suds. The amylases degrade the residues of starchy foods like porridge, potatoes, gravies, custard, chocolate, etc. to dextrins, while the lipases catalyse the hydrolysis of  salad oil, sauces, lipstick, etc. The cellulases in the detergents degrade mainly the the micr microfi ofibri brils ls which which are are gene genera rated ted durin during g cont continu inuou ouss use use and and repe repeate ated d washings of the garment and also help in restoring the original shine and colour  of the garment. The washing performance performance of the enzyme detergent detergent depends on many factors to achieve better results. These are detergent composition and dosage, pH and   buffer buffer capacit capacity y, wat water er hardness hardness,, was washin hing g time and tempera temperature ture,, mechan mechanical ical handling, handling, soiling agents, textile types to name a few. few. In addition, addition, the specificity specificity of the enzyme is another most important parameter. As a general opinion, it is cons conside idered red that that a deter deterge gent nt enzyme enzyme shoul should d have have as wi wide de a spec specifi ifici city ty as   possible. For example, a protease should be capable of degrading as many  proteins as possible. However, a reasonably good wash performance can be achieved by a specific protease, in comparison to a non-specific protease . As the hydro-lysis proceeds, small peptide fragments are formed by the action of an unspecific protease, which are rather difficult to remove as they are not very much soluble in detergent solutions. On the other hand, larger protein or peptide frag fragme ment ntss are are form formed ed on hydr hydrol olys ysis is wi with th a spec specif ific ic prot protea ease se due due to the the Page | 15  breakdown of very few peptide bonds which can be easily removed during the washing process. Presently, use of dual enzymes in detergent formulations is  practised, wherein the enzymatic hydrolysis and degradation can be broadened considerably in comparison to a single enzyme approach. Recently, workers of the Genencor International Inc., USA have developed enzyme enzymess cal called led endoglycosidases which which degly deglyco cosy sylat latee biop biopoly olyme mers rs like like glycoproteins which are widely distributed in living organisms. They employed rDNA technology to develop Endo-b -N-acetyl glucosaminidase H  ( Endo   Endo H ) as a clea cleani ning ng agen agent. t.   End has a uniq unique ue prop proper erty ty to remo remove ve bact bacter eria ia Endo o H  has (Staphylococci and E. coli) from glass and cloth surfaces in buffer and detergent solutions. At present, most of the advanced countries like Japan, United States and some Europe European an countr countries ies almost almost invariab invariably ly use the deterg detergent entss incorp incorporat orated ed wit with h enzymes. Interestingly, in Japan, all detergent brands contain enzymes. In India, a few premium detergent brands presently available in the market like  Ariel  (Pro (Procte cterr and and Ga Gamb mble le (Ind (India) ia) Ltd.) Ltd.),, Surf Surf Ultr Ultra, a, Rin Rin Biol Biolit ites es,, Reve Revell Plus Plus (Hin (Hindu dust stan an Leve Leverr Ltd. Ltd.)) and and  Zymo (Hen (Henke kel) l) cont contai ain n enzy enzyme mess in thei their  r  form formula ulati tions ons.. Rece Recentl ntly y, Proc Procter ter and and Ga Gamb mble le (P&G) (P&G) has intro introdu duce ced d a new new cellulase enzyme in the detergent powder,  Ariel , presently marketed in India, that eliminates the fuzz formed during washing and tumble-drying tumble-drying,, particularly particularly of the cotton fabrics. The manufacturers claim that use of this product retains the colour and improves the texture of the fabric on repeated washings. In dishwashing Enzymes have been successfully used in laundry detergents for many years as an aid to remove tough stains. However, the interest in using enzymes in automatic dishwashing detergents (ADDs) has increased recently. Both laundry and dishwashing detergents share similar functions such as removal of stains from egg, milk and starch-based soilings, etc. The performance of the enzymes in the ADDs is strongly strongly influenced influenced by the ADD formulation formulation and the conditions of the automatic dishwashing. At present, proteases and amylases are the only two enzymes which have found major application in dishwashing detergents. In  particular, enzyme-based dishwashing detergents are less abrasive in function and thus are suitable for use on delicate chinaware; they prevent the erosion of  designs and colours. This application application was first exploited in Japan where the use of richly decorated chinaware and wooden kitchen utensils is widespread. Enzymatic ADDs have gained widespread usage since the last decade. In the   pas pastt 2–3 2–3 year years, s, AD ADDs Ds wi with th enzym enzymes es we were re laun launch ched ed in seve severa rall Euro Europe pean an coun countri tries, es, viz. viz. Austr Austria ia,, Ge Germa rmany ny,, Sw Switz itzerl erlan and, d, De Denm nmark ark and and the the Unite United d Kingdom. In Japan, all major ADD brands contain enzymes, whereas only one  brand in the US market currently contains enzymes. However, at present, there are no enzymatic dishwashing detergents available in India. Page | 16 In the textile industry Currently Currently,, in the textile industry, industry, there is a widespread widespread demand for faded jeans. This This invol involve vess subje subjecti cting ng such such cloth clothes es to amyla amylase sess – a proc process ess commo commonl nly y referred to as biowashing  or  biobleaching , an alternative to the term, enzyme fade. This allows elegant softness and unique shades to be given to the cloth which overcomes the traditional methods of bleaching by sodium hypochlorite or tumb tumbli ling ng wi with th pumi pumice ce ston stones es,, and and also also offe offers rs bett better er safe safety ty as we well ll as economy. In food and dairy industries With the better understanding of such enzymes, more and more areas of their  application are emerging, such as in dairy, food and beverage industries. The use of enzymes in these industries in the cleaning operations helps in creating the required hygienic conditions in such plants. Probably, the use of enzyme based detergents in the in-place cleaning of membranes of ultrafiltration (UF) and reverse osmosis (RO) equipments proves promising and forms one of the most important aspects of modern dairy and food industries. The UF and RO membranes are put to a variety of uses including concentration, clarification and/or sterilization of liquid foods like skim milk, whey, egg white, fruit juices and beverages. Despite their diverse applications, these two membrane processes have some inherent disadvantages. The membrane filters come in contact with the feed stock during use. Even a small degree of adsorption causes pore blockage resulting resulting in clogging clogging of filters, a phenomenon called fouling, and thereby cause a reduction in the permeate flux rate and loss in the product quality with increase in production costs. In general, the proteins, inorganic salts and fat resid residues ues along along wi with th bacte bacteria ria cons constit titute ute the commo common n and and import importan antt foulin fouling g agents responsible for lowering the flux and affecting the product quality. Depending on the type of application, the precise formulations are made; for  inst instan ance ce,, prot protea ease sess are are used used for for foul fouled ed dair dairy y filt filter ers, s, a -amy -amyla lase sess and and b -glucanases in yeast and cereal, and cellulases and pectinases for wines and fruit  juices. The enzyme detergent preparations presently marketed for cleaning of  membra membrane ne systems systems are Terg-a-zyme (Alc (Alcon onox ox,, Inc, Inc, Ne New w York, ork, US USA) A) and and (Henke kell KG KGaA aA,, Dusse Dusseld ldorf orf,, Ge Germa rmany ny). ). Thes Thesee enzym enzymee-ba base sed d Ultras Ultrasil il 53 (Hen cleaners that have been marketed rely very much on the proteases to cleave and solubilize the protein foulant. The use of alkaline proteases from Bacillus sp. strain MK5-6 has also proved successful in our laboratory. Pilot scale evaluation of the enzymes at plant level operations for UF membrane cleaning indicated the the enzym enzymee prep prepar arat ation ion to be highl highly y effe effecti ctive ve and and resto restore red d 100% 100% flux flux in comparison to Terg-a-zyme, a commercial preparation which resulted in only 80% restoration of the flux. The use of proteases and lipases to degrade and solubilize protein and fat foulants has also proved beneficial. Page | 17 Other uses The The appli applica catio tion n of enzym enzymee-cle clean aner erss in the the opti optica call indu industr stry y is impor importan tant, t, enabling one to give 100% safe and efficient cleaning to lenses. In India,   pre prese sent ntly ly one one such such enzy enzyme me-b -bas ased ed opti optica call clea cleane nerr in the the form form of tabl tablet etss containing Subtilope Subtilopeptida ptidase se A is being marketed by M/s Bausch and Lomb (Indi (India) a) Ltd. Ltd. Enzy Enzyme me dete deterg rgen ents ts have have also also foun found d appl applica icati tion on in hosp hospita itals. ls.  Promod 153L, a protease enzyme-based cleaner, has been used to clean surgical instruments fouled by blood proteins. Benefits of enzymes The past decades with a growing number of enzyme applications in consumer  detergents have led to major improvements in terms of benefits for consumers. Low temperature efficiency . Enzy Enzyme mess ca cata taly lyze ze the the brea breakd kdow own n of soil soilss and and stai stain n mater ateria ials ls at lowe lower  r  temperatures. temperatures. This allowed washing at lower temperatures temperatures and using less water  throughout Europe whilst washing performance has improved. The energysaving in the home from the temperature temperature reduction and consequent reduction in environmental environmental emissions (such as carbon dioxide) is considerable as a washing machine operated at 40°C consumes only one third of the energy it would use at 95°C. Weight-efficiency Because enzymes act as catalysts (which can be used repeatedly to speed up chemical reactions without themselves being depleted) they are very weight efficient efficient and cost effective. effective. In other words, they can potentially replace a larger  larger  usage of conventional chemicals in the detergent. From an eco-toxicological viewpoint, enzymes can be considered as highly optimized laundry products ingredients which contribute positively to the overall environmental profile of  detergents. Other Technical and consumer research has demonstrated that the formulation of P&G detergents with enzyme has led to significant consumer benefits in terms of   performance. The benefits of enzymes are related to both the laundry process and the wash results, and include the abilities to: Page | 18 Wash at varying pH levels, from mild to high alkalinity; Use different wash temperatures, from 60°C to as low as the "30-40°C range"; Retain laundering performance in the presence of chemicals such as bleach;  builder, surfactant, etc…. Soften fabrics; Brighten their colors; Improve whiteness Remove fatty stains at low wash temperatures; Conclusions Thus to conclude, cleaning forms an important aspect for the maintenance of  hygiene and safety of foods in the food processing industry. Improperly cleaned food-contact surfaces lead to the accumulation of food particulates which favour  the formation of biofilms, i.e. attachment of microorganisms. These cause post contamination and spoilage of foods. It is, therefore, necessary to understand the interactions of the biotic and abiotic entities in the food-processing operations and further effectively analyse the impacts of cleaning and sanitation from a microbiological viewpoint. The use of enzyme-based detergents as biocleaners can also serve as a viable option to overcome the biofilm problem in the food industry. Further, the technology and production of these enzymes and the enzyme-based detergents is mostly patent-protected. As such most of the enzymes used in the detergent industry in India are being imported. Even the large scale detergent manu manufa fact ctur uree seem seemss high highly ly tech techni nica call requ requir irin ing g spec specif ific ic know know-h -how ow and and infrastructure. Work has been going on in the recent past in order to develop an indigenous technology on different enzyme systems in certain well reputed laboratories, laboratories, viz. National National Dairy Research Institute, Karnal, National National Chemical Chemical Laboratory, Pune and Institute of Microbial Technology, Chandigarh. Due to their high efficiency and safety, it is assumed that the enzyme detergents will eventually capture a bulk of the Indian detergent market. Page | 19 As in the other sectors of the chemical process industry, where enzymes are increas increasingl ingly y playing playing a crucia cruciall role in making making convent conventiona ionall process processes es more more envir environm onmen ent-f t-frie riend ndly ly,, the dete deterg rgen ents ts indu industr stry y has has also also benef benefit ited ed from from the the introduction of enzymes. The enzyme detergents are proving to be better than the traditional detergents with respect to washing performance, but there are still few constraints like the inability of the enzymes to withstand high alkalinity and variable temperatures. 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